RESEARCH ARTICLE TEADs, Yap, Taz, Vgll4s transcription factors control the establishment of Left- Right asymmetry in zebrafish Jonathan Fillatre1, Jean-Daniel Fauny2,3, Jasmine Alexandra Fels1, Cheng Li4, Mary Goll4, Christine Thisse1,2, Bernard Thisse1,2* 1Department of Cell Biology, University of Virginia, Charlottesville, United States; 2Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ Universite´ de Strasbourg, Illkirch-Graffenstaden, France; 3Institut de Biologie Mole´culaire et Cellulaire, Strasbourg, France; 4Department of Genetics, University of Georgia, Athens, United States Abstract In many vertebrates, establishment of Left-Right (LR) asymmetry results from the activity of a ciliated organ functioning as the LR Organizer (LRO). While regulation of the formation of this structure by major signaling pathways has been described, the transcriptional control of LRO formation is poorly understood. Using the zebrafish model, we show that the transcription factors and cofactors mediating or regulating the transcriptional outcome of the Hippo signaling pathway play a pivotal role in controlling the expression of genes essential to the formation of the LRO including ligands and receptors of signaling pathways involved in this process and most genes required for motile ciliogenesis. Moreover, the transcription cofactor, Vgll4l regulates epigenetic programming in LRO progenitors by controlling the expression of writers and readers of DNA methylation marks. Altogether, our study uncovers a novel and essential role for the transcriptional effectors and regulators of the Hippo pathway in establishing LR asymmetry. DOI: https://doi.org/10.7554/eLife.45241.001 *For correspondence: [email protected] Competing interests: The Introduction authors declare that no Formation of organs during embryonic development requires a progressive restriction of lineage competing interests exist. potential. This process, controlled by major signaling pathways, is achieved through changes in chro- Funding: See page 20 matin and in transcription factor (TF) networks: genes associated with pluripotency are progressively Received: 16 January 2019 silenced by DNA methylation, histone modifications and chromatin compaction while key TFs selec- Accepted: 11 September 2019 tively activate the expression of tissue specific genes (Boland et al., 2014; Reik, 2007). Therefore, Published: 12 September 2019 knowing how the activity of TFs and epigenetic modification of the chromatin control organogenesis in vivo is essential to our understanding of both normal development and diseases. As a model sys- Reviewing editor: Julien tem for organogenesis, we use the formation of the Kupffer’s vesicle (KV), the first organ formed in Vermot, Institut de Ge´ne´tique et de Biologie Mole´culaire et the zebrafish embryo and that functions as the Left-Right Organizer (LRO). The KV is the fish homo- Cellulaire, France log of the ventral node of the mouse, the Xenopus gastrocoel roof plate and the notochordal plate in rabbit. This organ is composed of ~50 monociliated cells organized as a hollow sphere with motile Copyright Fillatre et al. This cilia facing its lumen. Rotation of these cilia generates a transient counterclockwise fluid flow that article is distributed under the directs asymmetric activation of a conserved Nodal signaling pathway that guides asymmetric mor- terms of the Creative Commons Attribution License, which phogenesis of developing organs (Dasgupta and Amack, 2016). This vesicle derives from a small permits unrestricted use and population of ~20 precursor cells called the dorsal forerunner cells (DFCs), which are specified at the redistribution provided that the dorsal margin of the embryo at the onset of gastrulation in response to Nodal signaling original author and source are (Essner et al., 2005; Oteiza et al., 2008). During gastrulation, DFCs arrange into a cluster that credited. undergoes progressive compaction, followed by a mesenchymal to epithelial transition and Fillatre et al. eLife 2019;8:e45241. DOI: https://doi.org/10.7554/eLife.45241 1 of 25 Research article Developmental Biology organization of a single rosette. Following rosette formation, the center of this rosette opens to pro- gressively give rise to the lumen of the differentiated KV. Finally, ciliogenesis takes place during the last phases of differentiation of DFCs into the KV. Altogether, the epithelial organization of KV pro- genitors associated with both luminogenesis and ciliogenesis leads to the formation of a functional LRO (Matsui and Bessho, 2012). The regulation of the organogenesis of the LRO, from the specification of its progenitors to a fully functional KV, is well described and involves the activity of Nodal, FGF, non-canonical Wnt, Notch and Hedgehog signaling pathways (Matsui and Bessho, 2012). Conversely, a very limited number of TFs expressed in DFCs has been implicated in this process. Six genes have been identified so far, two Sox TFs: Sox32 and Sox17; two T-box TFs: Tbxta (also known as notail) and Tbx16 (also known as spadetail) and two TFs required for ciliogenesis: Foxj1a and Rfx2 (Aamar and Dawid, 2010; Amack and Yost, 2004; Bisgrove et al., 2012; Kikuchi et al., 2001; Yu et al., 2008). It is highly unlikely that these six TFs are the only transcriptional regulators of the developmental program lead- ing to the formation of the LRO. Indeed, in this study, we identified six additional transcription fac- tors (TFs) and/or cofactors (TcoFs) crucial for the formation and function of the KV. Strikingly, although the Hippo signaling pathway was previously identified as a major regulator of tissue growth and organ size (Johnson and Halder, 2014; Zhao et al., 2011), we discovered that the DNA binding TFs (Tead1a and Tead3a), the TcoFs mediators of the Hippo signaling pathway (Yap and Taz) as well as the TcoFs Vgll4b and Vgll4l (two factors homologous to the mammalian Vgll4 that negatively reg- ulates the activity of Yap and Taz) are upstream regulators of the formation and function of the LRO. These TFs and TcoFs (collectively named Hippo TFs/TcoFs thereafter in the text) control the function of signaling pathways involved in this process as well as the expression of genes essential to the for- mation and function of a ciliated epithelium with motile cilia. Finally, we identified that Vgll4l controls the expression in LRO progenitors of epigenetic factors, writers (the de novo DNA methyltransfer- ases) and readers (Methyl-CpG binding domain proteins) of DNA methylation marks that we found essential for DFCs proliferation and survival as well as for the formation of motile cilia. Results Hippo TFs/TcoFs are required for the establishment of LR asymmetry To identify novel factors involved in the transcriptional regulation of the formation of the LRO we screened the zebrafish gene expression patterns database (http://zfin.org/) for TFs or TcoFs specifi- cally expressed in the KV and/or in its progenitors. By this approach, we identified Vgll4l, a TcoF member of the Vestigial like four family, which is strongly expressed at gastrula stage in LRO pro- genitors (Figure 1A). Vestigial like family members are TcoFs known to function mainly through the interaction with TEA domain DNA-binding family of transcription factors (TEAD) (Deng and Fang, 2018). TEADs are the DNA binding TFs to which the TcoFs that mediate the transcriptional outcome of the Hippo sig- naling pathway, Yap and Taz (also known as Wwtr1), bind to, to activate expression of their target genes. Interestingly, in various human cancer cell lines, Vgll4 was shown to negatively regulate the transcriptional outcome of Hippo signaling by competing with Yap and Taz for TEADs, therefore inhibiting their function (Zhang et al., 2014). In addition to Yap and Taz, the zebrafish genome codes for three members of the Vgll4 family (Vgll4a, Vgll4b, Vgll4l) and four TEADs (Tead1a, Tead1b, Tead3a, Tead3b). However, only Vgll4l, Vgll4b, Yap, Taz, Tead1a and Tead3a are expressed in KV and/or KV progenitors (Figure 1A, Fig- ure 1—figure supplement 1). To investigate the function of these TFs and TcoFs in the formation of the LRO we performed general and/or DFC specific (Amack and Yost, 2004) knockdown experi- ments using translation interfering and/or splice interfering morpholinos (MOs) and analyzed the effect of these loss of functions on the establishment of the LR asymmetry of the embryo. Looking at the direction of the heart jog at one day of development (Figure 1B) and at the expression of lefty1 in dorsal diencephalon (Thisse and Thisse, 1999) at 20 hr post fertilization (Figure 1—figure supple- ment 2) we found that loss of function of each of these TFs or TcoFs strongly disrupts embryo later- ality. Specificity of knockdown phenotypes was demonstrated by their reproducibility using different non overlapping MOs and in rescue experiments through injection of in vitro synthesized, MOs insensitive, mRNAs (Figure 1B). The implication of Yap in embryo laterality was further confirmed Fillatre et al. eLife 2019;8:e45241. DOI: https://doi.org/10.7554/eLife.45241 2 of 25 Research article Developmental Biology Figure 1. Hippo TFs/TcoFs are essential for establishing the left-right asymmetry. (A) Whole-mount in situ hybridization for vgll4l, yap, taz, vgll4b and tead3a at gastrula and at the 6-somite stage. Vgll4l is expressed in DFCs at 60% (dorsal view), 80% (lateral view) and 90% epiboly (vegetal pole view) but is not expressed in the KV at the 6-somite stage (vegetal pole view).